Organisms such as E. coli that inhabit the human gut tract enjoy the advantages of an abundant supply of nutrients, but there are also special challenges. For example, they must be able to tolerate high concentrations of bile salts (e. g., chenodeoxycholate derivatives), which are secreted into the intestine to aid in the digestion and absorption of fats. The detergent properties of bile salts make them toxic to microbes because of their solubilizing action on cell membranes. Members of the intestinal microbiota have evolved several mechanisms to protect themselves from these effects. On a practical note, laboratory growth media that contain bile salts, such as MacConkey agar, are used to selectively cultivate enteric organisms

1. Suppose you generated a mutant strain of E. coli that cannot grow in the presence of bile salts. You suspect the defect lies in either (1) the cell’s lipopolysaccharide or (2) transporters that export bile salts from the cell. To investigate the first possibility, you decide to use cell fractionation to obtain material present in the cell's

2. Biochemical analysis of the mutant cell's lipopolysaccharide does not reveal any differences that might explain the cell's sensitivity to bile salts. You, therefore, extend your investigation to possible defects in the AcrB protein. This transporter harnesses the cell's proton motive force to export a variety of drugs. Before you can analyze this protein, you will need to purify it. Using the same fractionation technique as before, you begin by attempting to isolate the cell's

3. Success! You have determined that the responsible efflux pump in your mutant is unable to bind chenodeoxycholate. Now you are ready to identify the genetic basis for this defect, so you proceed to isolate the DNA from the fraction of your preparation.